Project Summary/Abstract Adenoid Cystic Carcinoma (ACC) is the second most frequent malignancy of the minor and major salivary glands and has poor long-term prognosis. Molecular studies of ACC tumors have been complicated by the relative rarity of the tumors, differences in diagnosis and characterization, and the lack of bona fide ACC cell lines. A large majority of ACC tumors contain a recurrent t(6;9) translocation which fuses the MYB proto- oncogene on chromosome 6q to the NFIB gene on chromosome 9p. The translocations have multiple effects: leading to the expression of truncated, oncogenic forms of the c-Myb transcription factor and juxtaposing the MYB gene next to tissue specific enhancers that lead to overexpression in ACC tumors. The major challenge in studying ACC tumors has been the need to perform detailed molecular characterizations on rare tumor samples that are old enough to have clinical outcome information. To address that challenge, we developed and optimized innovative RNA-seq methods to analyze RNA derived from archival Formaldehyde-Fixed, Paraffin-Embedded (FFPE) ACC tumor samples, which allowed us to perform in-depth transcriptome analyses on these rare tumor samples. Our efforts led to the identification of novel, recurrent fusions involving both MYB and the related MYBL1 oncogene, which encodes the A-Myb transcription factor. We uncovered new insights into the mechanisms of activation of these genes in ACC tumors and characterized the gene expression profiles of ACC tumors and how they are related to MYB and MYBL1 oncogene expression. These findings put us in a unique position to investigate the mechanisms leading to ACC tumors and to identify the important regulators and potential therapeutic targets in ACC tumors. Our results lead us to hypothesize that salivary gland ACC tumors are caused by mutated MYB or MYBL1 oncogenes overexpressed due to regulation by tissue-specific enhancers, resulting in the induction of a characteristic, ACC-specific gene expression program. In this revised renewal application, we will build on the studies that we have completed and make use of the extensive RNA-seq data that we have generated for ACC tumor samples. We will focus on performing extensive and in-depth bioinformatics analyses of the RNA-seq data and on performing molecular validations to gain insights into the mechanisms that lead to ACC tumors. We will also investigate the mechanisms that lead to overexpression of the MYB and MYBL1 oncogenes in ACC tumors, with the goal of identifying new therapeutic targets. We have assembled an interactive team of investigators with expertise in molecular biology, genomics, bioinformatics, biostatistics and computational methods who will focus on these aims to answer key questions about the biology of these devastating tumors.